Tyrosine hydroxylase (TH) catalyzes the hydroxylation of tyrosine to dihydrophenylalanine in the rate limiting step of catecholamine biosynthesis. The reaction requires the reduction of molecular oxygen to water with an oxygen atom incorporated into the aromatic ring of tyrosine. TH is dependent on tetrahydrobiopterin as well as a non-heme iron for activity. Current evidence for both the phenylalanine hydroxylase (PAH) and TH systems suggests that a hydroperoxytetrahydrobiopterin is the hydroxylating species. The role of the iron is unclear; however, the lack of activity in iron-free TH is consistent with the Fe having an essential role in catalysis. The Fe is proposed to facilitate the formation of the peroxy intermediate while an Fe oxo intermediate as the hydroxylating species has not been ruled out. Present studies are focused on the identification of the hydroxylating species of the TH reaction and the stoichiometry and order of binding of the reactants. Preliminary results have been obtained by monitoring the reaction in a sealed chamber with an oxygen electrode. In air-saturated buffer one mole of molecular oxygen is consumed for each mole of TH active sites in the absence of tyrosine. This oxygen uptake appears to be dependent on tetrahydrobiopterin and not tyrosine, evidence that the oxygen dependent formation of the hydroxylating species may be separate from the tyrosine dependent hydroxylation. Further studies are underway to confirm these results as well as to isolate the oxygen activation from the hydroxylation steps into separate aerobic and anaerobic reactions. - tyrosine hydroxylase, catecholamine, dihydrophenylalanine, tetrahydrobiopterin, intermediate